The instant disclosure pertains to a method for anchoring oligonucleotides containing multiple reaction sites to a substrate. In particular, the instant disclosure pertains to a method for anchoring a hairpin-shaped oligonucleotide containing a multiple site thiophosphate backbone to a glass substrate. The instant disclosure also pertains to such an oligonucleotide, as well as a probe and a kit containing the same.
Immobilization of probe or target molecules to a solid substrate is an integral part of many bio-assay techniques. Glass substrates are generally preferred over polymer substrates for a variety of reasons. Glass substrates possess increased tolerance to higher temperatures and higher ionic strength washes. Unlike polymer substrates which are porous, nonporous glass substrates retain less extraneous material which could contribute to background signals. Furthermore, because glass is non-porous, volumes of biomolecules spotted onto the surface of a glass substrate can be kept to a minimum. Glass has low background fluorescence which does not interfere appreciably with the sensitivity of fluorescence-based detection methods. Also, the silanol surface of glass can be readily modified by the silanization of functional silanes.
In certain applications, it is preferable to covalently attach oligonucleotides and DNA samples to glass surfaces. Many of the methods presently used for covalently attaching oligonucleotides to glass surfaces employ bifimctional linkers or biotin-streptavidin linkages. For example, in Hybridization of DNA Targets to Glass-Tethered Oligonucleotide Probes, 4 MOLECULAR BIOTECHNOLOGY 213 (1995), Wanda G. Beattie et al. disclose the attachment of surface oligonucleotides by linking a 5xe2x80x2 terminal amine to an epoxysilanized glass. The attachment reaction takes place in 100 mM NaOH, preferably. Small volumes ( greater than 1 nL) of reaction solution are dispensed or xe2x80x9cspottedxe2x80x9d onto the substrate surface and evaporate very quickly. Because the dispensed liquid evaporates so quickly, chemical reactions must be fast and efficient in order that a sufficient number of molecules attach within a brief time period. Furthermore, the alkali concentration of the reaction solution increases as the solvent evaporates, causing oligonucleotide degradation, which in turn contributes to false signals and high background in the assay. Elevated alkali concentration may also cause the silane to detach from the glass substrate.
Michael Pirrung and coworkers at Duke University have discovered that oligonucleotides with a 5xe2x80x2-phosphothioate react with bromoacetamide derivatized silane on a glass surface through nucleophilic substitution. The reaction takes approximately one half hour and is performed at neutral pH in aqueous solution. Unfortunately, 5xe2x80x2-phosphorylation is expensive, particularly for large numbers of oligomers, and any unsuccessful incorporation followed by sulfurization is not effective for attachment.
Simply increasing the concentration of probes or DNA primers to enhance the reaction rate of attachment cannot solve the dilemma of attaching oligonucleotides to surfaces. For example, the surface area of a slide is limited. The highest density of oligonucleotides on a glass surface is 0.1 pmole/mm2 which is equivalent to one molecule every 39 square angstroms (Southern, et al., Nature Genetics Supplement, 21, 5-9 (1999)). The accessibility of oligonucleotides to the surface area (0.013 mm2, 0.57 nL) of a dispensed drop in 130 xcexcm diameter is saturated at 2.3 xcexcM (1.33 fmole). Moreover, it is questionable whether densely packed substrates provide a solution, as densely packed substrates have been observed to reduce enzyme accessibility.
As the above discussion suggests, improvements are still possible and desirable in the area of covalent linking of oligonucleotides to surfaces, especially glass surfaces. For example, a method is needed for covalently linking oligonucleotides to glass surfaces which exhibits improved reliability and reproducibility. In particular, a method is needed which allows better control of reaction efficiency, molecular density and conformation, and enzyme compatibility. Ideally, such a method would also be economical to use. These and other concerns are addressed in greater detail below.
The instant disclosure pertains to a method for attaching an oligonucleotide primer to a substrate that comprises attaching a reactive group onto a substrate, and reacting the substrate with an oligonucleotide primer containing multiple reactive groups corresponding to the reactive group contained on the substrate to bind the oligonucleotide primer to the glass substrate.
The substrate can be glass, inorganic or organic polymer, and metal. The reactive group of the substrate can contain electrophilic Cxe2x95x90C double bonds for a nucleophilic addition, or disulfide for disulfide exchange. For example, the reactive group of the substrate can be maleimide and vinylsulfonate, benzene sulfonate, methanesulfonate, and trifluoromethanesulfonate.
The multiple reactive groups contained on the oligonucleotide primer can be aminoalkyl, sulfhydryl, and thiophosphate groups. The oligonucleotide primer can contain multiple reactive groups that are the same, or it can contain at least two different reactive groups. The reactive groups contained on the oligonucleotide primer can be adjacent one another or distributed randomly throughout the primer. The oligonucleotide primer may also contain a nucleotide sequence corresponding to a protein binding site. The oligonucleotide primer may be in the configuration of a hairpin having a loop containing multiple reactive sites.
In particular, a method for attaching an oligonucleotide primer to a glass substrate is disclosed that comprises preparing a bromoacetamide derivatized silane glass surface on a glass substrate, and reacting the bromoacetamide derivatized silane glass surface with an oligonucleotide primer containing multiple thiophosphate groups to bind the oligonucleotide primer to the glass substrate.
The instant disclosure also pertains to an oligonucleotide primer comprising a backbone having multiple reactive groups for attaching said oligonucleotide primer to a substrate.
The instant disclosure also pertains to an oligonucleotide primer bound to a substrate according to the method described above.
The instant disclosure also pertains to a bioassay kit comprising an oligonucleotide primer bound to a substrate as described above.
The instant disclosure also pertains to a method for immobilizing a PCR product to a substrate that comprises creating an oligonucleotide primer by phosphorylating the 5xe2x80x2 end of the oligonucleotide primer having multiple reaction sites and modifying the ""3 end of said oligonucleotide primer to include the PCR primer sequence, hybridizing the single-stranded 3xe2x80x2 end of said oligonucleotide primer with the PCR product, and subjecting reaction product to enzymatic or chemical ligation.